Cover Page The handle http://hdl.handle.net/1887/62615 holds various files of this Leiden University dissertation. Author: Xia, L. Title: Corpora non agunt nisi fixata : ligand receptor binding kinetics in G protein- coupled receptors Issue Date: 2018-05-30
Cover Page
The handle http://hdl.handle.net/1887/62615 holds various files of
this Leiden University dissertation. Author: Xia, L. Title: Corpora
non agunt nisi fixata : ligand receptor binding kinetics in G
protein- coupled receptors Issue Date: 2018-05-30
Receptor Antagonists
Lizi Xia, Wessel A. C. Burger, Jacobus P.D. van Veldhoven, Boaz J.
Kuiper, Tirsa T. van Duijl,
Eelke B. Lenselink, Ellen Paasman, Laura H. Heitman, and Adriaan P.
IJzerman
Adapted from: J. Med. Chem., 2017, 60(17): 7555–7568
120
About this chapter
We expanded on a series of pyrido[2,1-f]purine-2,4-dione
derivatives as human adenosine A3
receptor (hA3R) antagonists to determine their kinetic profiles and
affinities. Many compounds
showed high affinities and a diverse range of kinetic profiles. We
found hA3R antagonists with very
short residence time (RT) at the receptor (2.2 min for 5) and much
longer RTs (e.g. 376 min for 27, or
391 min for 31). Two representative antagonists (5 and 27) were
tested in [35S]GTPγS binding assays,
and their RTs appeared correlated to their (in)surmountable
antagonism. From a kon-koff-KD kinetic
map we divided the antagonists into three subgroups, providing a
possible direction for the further
development of hA3R antagonists. Additionally, we performed a
computational modelling study that
sheds light on the crucial receptor interactions dictating the
compounds’ binding kinetics. Knowledge
of target binding kinetics appears useful for developing and
triaging new hA3R antagonists in the
early phase of drug discovery.
121
Introduction
The adenosine A3 receptor is the youngest member discovered in the
family of adenosine receptors,1
all of which belong to class A G-protein coupled receptors (GPCR)
and fall into four distinct subtypes
(A1, A2A, A2B and A3). Although all subtypes are activated by the
endogenous ligand adenosine, these
purinergic receptors differ from each other in their distribution
and to which G protein they are
coupled. Following agonist activation, the A1 and A3 adenosine
receptors cause a decrease in cAMP
levels as they primarily couple to Gi proteins. The A2A and A2B
adenosine receptors on the other hand,
are primarily linked to Gs proteins and this leads to increased
levels of cAMP upon receptor
activation.2
Although the pharmacological characterization of adenosine
receptors has been well documented,3
the human adenosine A3 receptor (hA3R) is less well characterized
because of its “dichotomy” in
different therapeutic applications.4 Moreover, certain ligands have
been described as cytoprotective
or cytotoxic merely depending on the concentration employed,
highlighting the difficulties that arise
when characterizing novel hA3R compounds.5 Nevertheless, there is
no doubt that the hA3R has
therapeutic potential in clinical indications (i.e. cardiovascular
diseases,6, 7 cancer,7, 8 and respiratory
diseases7, 9-11), due to its overexpression on cancer and
inflammatory cells.3, 12-15
Traditional drug screening methods, and those employed in previous
hA3R drug discovery attempts,
revolve around the use of a ligand’s affinity as the selection
criterion for further optimization in a so-
called structure-affinity relationships (SAR) approach. In recent
years, however, there has been
emerging the realization that selecting ligands based on their
affinity, an equilibrium parameter, does
not necessarily predict in vivo efficacy. This is due to the
dynamic conditions in vivo, that often are in
contrast to the equilibrium conditions applied in in vitro
assays.16 In fact a ligand’s kinetic properties
may provide a better indication of how a ligand will perform in
vivo.17 Specifically, the parameter of
residence time (RT) has been proposed as a more relevant selecting
criterion. The RT reflects the
122
lifetime of the ligand-receptor complex and can be calculated as
the reciprocal of the ligand’s
dissociation constant (RT=1/koff).18, 19
Table 1. Binding Affinity and Kinetic Parameters of
1-benzyl-8-methoxy-3-propylpyrido[2,1-f]purine-
2,4(1H,3H)-dione23, 24
a pKi ± SEM (n ≥ 3, average Ki value in nM), obtained at 25 oC from
radioligand binding assays with [3H]PSB-11
([3H]34) on human Adenosine A3 receptors stably expressed on CHO
cell membranes.
b KRI (n = 2, individual estimates in parentheses), obtained at 10
oC from dual-point competition association
assays with [3H]34 on human Adenosine A3 receptors stably expressed
on CHO cell membranes.
c kon ± SEM (n ≥ 3), obtained at 10 oC from competition association
assays with [3H]34 on human Adenosine A3
receptors stably expressed on CHO cell membranes.
d koff ± SEM (n ≥ 3), obtained at 10 oC from competition
association assays with [3H]34 on human Adenosine A3
receptors stably expressed on CHO cell membranes.
e RT (min) = 1/(60*koff).
While the binding kinetics of some (labeled) hA3R agonists have
been studied,20 this parameter has
not been part of medicinal chemistry efforts for antagonists, i.e.
yielding structure-kinetics
relationships (SKR), next to SAR.21 Therefore, in order to provide
the first SKR analysis on the hA3R, a
highly potent and selective hA3R antagonist scaffold was chosen.
The pyrido[2,1-f]purine-2,4-dione
template has been previously characterized with respect to affinity
alone. In a Topliss approach22 we
had synthesized and characterized a number of highly potent and
selective hA3R antagonists.23, 24
One of the reference antagonists (1) with good affinity and
selectivity over other adenosine
receptors is represented in Table 1. Using this compound as the
starting point, we further selected
and synthesized compounds to add to the library of
pyrido[2,1-f]purine-2,4-dione derivatives. Using
Compound pKi
a ± SEM
c (M-1 s-1) koff d
(s-1) RT e (min)
1 8.5 ± 0.02 (3.2) 0.99 (0.97; 1.0) (8.5 ± 1.2) x 105 (3.2 ± 0.02)
x 10-4 52 ± 0.3
123
radioligand displacement assays and competition association assays,
we obtained affinity (Ki) and
kinetic parameters (kon, koff, and RTs). This allowed a full SKR
study alongside a more traditional SAR
analysis. The findings provide information on the structural
requirements for a favorable kinetic
profile at the hA3R and consequently may improve the in vitro to in
vivo translation for hA3R
antagonists.
Chemistry.
The synthesis approach shown in Scheme 1 was adapted from Priego et
al.23, 24 Starting from the
commercially available materials benzylurea (3), ethyl cyanoacetate
and sodium methoxide 1-benzyl-
6-amino-uracil (4) was synthesized in a 88% yield.25 In situ
dibromination of uracil 4 at the C5 position
by N-bromosuccinimide, followed by cyclisation with
4-methoxypyridine gave the pyrido[2,1-
f]purine-2,4-dione (5) in a one pot reaction. Final compounds 1, 2
and 6-22 (as depicted in Table 1)
were obtained, with yields varying in the range of 3-86%, by
alkylating the N3 position of 5 using a
variety of alkyl, alkenyl and alkynyl bromides in acetonitrile and
1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) as a base. Secondly, to be able to diversify on the N1 (R2)
position, building block 23 had to be
obtained. Full conversion of methylcyclopropyl compound 2 into the
desired debenzylated 23 was
realized by multiple additions of ammonium formate and Pd(OH)2 at
80 oC in ethanol overnight. Due
to poor solubility 23 was extracted with hot DMF and Pd(OH)2 was
removed by filtration, resulting in
a quantitative yield. Finally various N1 substituted benzyl (24-32)
and phenethyl (33) derivatives
(Scheme 1) were made starting from the respective benzyl- or
phenethyl bromides in DMF with
K2CO3 used as base.
All binding affinities of the pyrido[2,1-f]purine-2,4-dione
derivatives were determined at 25 oC in a 2
hours incubation protocol. All compounds were able to
concentration-dependently inhibit specific
124
[3H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-
imidazo[2,1-i]-purin-5-one26 ([3H]PSB-11,
34) binding to the human adenosine A3 receptor and their affinities
are listed in Tables 1, 2 and 3. All
compounds had (sub)nanomolar binding affinities ranging from 0.38
nM for compound 27 to 108 nM
for compound 5.
Scheme 1. Synthesis of
1,3-disubstituted-1H,3H-pyrido[2,1-f]purine-2,4-dione
derivatives.
Reagents and conditions: a) ethyl cyanoacetate, NaOEt, EtOH,
reflux, overnight; b) i) NBS, CH3CN, 80 oC, 1h ii)
4-methoxypyridine, 80 oC, overnight; c) R1-Br, DBU, CH3CN, 80 oC,
overnight; d) 20% Pd(OH)2, ammonium formate, EtOH, reflux,
overnight; e) R2-Br, K2CO3, DMF, 40 oC overnight.
Subsequently, the human adenosine A3 receptor ligands were screened
in a so-called “dual-point”
competition association assay,27 allowing for the semi-quantitative
estimation of the compounds’
dissociation rates and therefore the compounds’ RTs. The specific
binding of [3H]34 was measured
after 20 and 240 minutes in the absence and presence of a single
concentration (i.e. 1 x IC50) of
unlabeled human adenosine A3 receptor antagonists, which yielded
their Kinetic Rate Index (KRI). A
long RT compound shows a characteristic “overshoot” followed by a
steady decrease in specific
binding until a new equilibrium is reached; in such a case the KRI
value is greater than unity.
Conversely, a ligand with a fast dissociation rate is represented
by a more shallow curve, yielding a
KRI value smaller than one when dividing the binding at t1 by the
binding at t2. The KRI values in the
series ranged from 0.38 to 4.06 (Table 1, 2 and 3).
125
Table 2. Binding Affinities and Kinetic Parameters of
Pyrido[2,1-f]purine-2,4-dione Derivatives with
Modification on N-3 Position (R1 group)
Compd. R1
7 CH2CH3 8.0 ± 0.1
8 CH2CH2CH2CH3 8.8 ± 0.1
9 CH2CH2CH2CH2CH3 8.5 ± 0.02
106 (6.0 ± 0.5) x
105 (8.2 ± 1.3) x
105 (6.2 ± 0.2) x
(5.9) 0.72
(4.3) 1.20
(1.4) 1.23
15 CH2CH2OCH3 7.7 ± 0.2
105 (6.3 ± 0.7) x
17 CH2CH(CH3)2 8.9 ± 0.02
20 CH2CH2C(CH3)3 8.1 ± 0.02
21 CH2Si(CH3)3 8.6 ± 0.03
2 CH2C3H5 9.0 ± 0.02
126
a pKi ± SEM (n ≥ 3, average Ki value in nM), obtained at 25 oC from
radioligand binding assays with [3H]34 on
human Adenosine A3 receptors stably expressed on CHO cell
membranes.
b KRI ± SEM (n = 3) or KRI (n = 2, individual estimates in
parentheses), obtained at 10 oC from dual-point
competition association assays with [3H]34 on human Adenosine A3
receptors stably expressed on CHO cell
membranes.
c kon ± SEM (n ≥ 3), obtained at 10 oC from competition association
assays with [3H]34 on human Adenosine A3
receptors stably expressed on CHO cell membranes.
d koff ± SEM (n ≥ 3), obtained at 10 oC from competition
association assays with [3H]34 on human Adenosine A3
receptors stably expressed on CHO cell membranes.
e RT (min) = 1/(60*koff).
f n.d. = not determined.
Compounds with a KRI value less than 0.7 or greater than 1.5 were
selected for complete kinetic
characterization through the use of a competition association assay
with [3H]34 (Figure 1A). In order
to obtain extensive Structure-Kinetics Relationships (SKR), close
structural analogs ( 9, 28, 29 and 30)
of 1 were also tested to obtain their association (kon) and
dissociation (koff) rate constants.
Association rate constants varied by 30-fold, ranging from (1.0 ±
0.1) x 105 M-1s-1 for antagonist 31 to
(3.0 ± 0.3) x 106 M-1s-1 for antagonist 30 (Table 3).
Interestingly, there was an approximately 290-fold difference in
dissociation rate constants,
reflecting the divergent KRI values. Antagonist 5 had the fastest
dissociation rate constant of (1.4 ±
0.5) x 10-2 s-1 and thus the shortest RT of 2.2 min, while both
antagonist 27 and 31 had the slowest
dissociation rate constants of (4.7 ± 0.7) x 10-5 s-1 and (5.3 ±
1.5) x 10-5 s-1, respectively, and thus the
longest RTs of 376 min and 391 min, respectively. Of note, the long
RT antagonist 27 (Figure 1A)
127
Table 3. Binding Affinities and Kinetic Parameters of
Pyrido[2,1-f]purine-2,4-dione Derivatives with
Modification at R2
Compd. R2 pKi
c (M-1 s-1) koff d
(s-1) RT e (min)
2 benzyl 9.0 ± 0.02
(1.0) 2.68 ± 0.48 (2.8 ± 0.5) x 106 (6.0 ± 1.7) x 10-5 315 ±
105
24 3-CH3-benzyl 8.8 ± 0.02
25 4-CH3-benzyl 9.0 ± 0.1
26 4-CH2CH3-benzyl 9.2 ± 0.04
27 3-OCH3-benzyl 9.4 ± 0.03
(0.38) 2.24
(2.32; 2.15) (4.8 ± 0.2) x 105 (4.7 ± 0.7)x 10-5 376 ± 58
28 4-OCH3-benzyl 8.9 ± 0.01
(1.4) 1.39
(1.22; 1.55) (4.8 ± 0.1) x 105 (7.8 ± 2.0) x 10-5 250 ± 72
29 3-Cl-benzyl 8.3 ± 0.02
(4.9) 0.89
(1.06; 0.72) (8.2 ± 1.3) x 105 (4.7 ± 0.7) x 10-4 36 ± 5.5
30 4-Cl-benzyl 8.9 ± 0.01
(1.2) 1.11
(1.02; 1.20) (3.0 ± 0.3) x 106 (8.2 ± 0.2) x 10-4 20 ± 0.5
31 3,4-dichlorobenzyl 8.3 ± 0.01
(5.3) 3.12
(3.49; 2.75) (1.0 ± 0.1) x 105 (5.3 ± 1.5) x 10-5 391 ± 137
32 4-Br-benzyl 8.9 ± 0.1
33 phenethyl 8.1 ± 0.04
(1.21; 0.97) n.d. n.d. n.d.
a pKi ± SEM (n ≥ 3, average Ki value in nM), obtained at 25 oC from
radioligand binding assays with [3H]34 on
human Adenosine A3 receptors stably expressed on CHO cell
membranes.
b KRI ± SEM (n = 3) or KRI (n = 2, individual estimates in
parentheses), obtained at 10 oC from dual-point
competition association assays with [3H]34 on human Adenosine A3
receptors stably expressed on CHO cell
membranes.
c kon ± SEM (n ≥ 3), obtained at 10 oC from competition association
assays with [3H]34 on human Adenosine A3
receptors stably expressed on CHO cell membranes.
d koff ± SEM (n ≥ 3), obtained at 10 oC from competition
association assays with [3H]34 on human Adenosine A3
receptors stably expressed on CHO cell membranes.
e RT (min) = 1/(60*koff)
f n.d. = not determined.
128
Figure 1: (A) Representative competition association assay curves
of [3H]34 in the absence (control) or
presence of a long residence time compound 27 and a short residence
time compound 5. Experiments were
performed at 10 °C using the compound’s respective IC50 value at
the hA3R. (B) Competition association curves
of [3H]34 in the absence (control) or presence of long residence
time compound 27. Experiments were
performed at 25 °C using the compound’s respective IC50 value at
the hA3R. t1 is the radioligand binding at 20
min, while t2 is the radioligand binding at 240 min.
displayed a typical “overshoot” in the competition association
curve, indicative of a slower
dissociation than the radiolabeled probe [3H]34, while the short RT
antagonists, exemplified by
antagonist 5 (Figure 1A) presented more shallow, gradually
ascending curves. There was a good
correlation between the negative logarithm of the antagonists’
dissociation rate constants and their
KRI values derived from the kinetic screen (Figure 2A), which
confirmed that a compound’s KRI value
A.
B.
3 0 6 0 9 0 1 2 0 1 5 0 1 8 0 2 1 0 2 4 0 -2 0
0
[ 3
+ c o m p o u n d 2 7
+ c o m p o u n d 5
t 1 t2
3 0 6 0 9 0 1 2 0 1 5 0 1 8 0 2 1 0 2 4 0 -2 0
0
[ 3
+ c o m p o u n d 2 7
t 1 t 2
129
is a good predictor for its dissociation rate constant. Notably,
the experimental temperatures in the
kinetic assays were lower than in the equilibrium displacement
assays (25 oC vs 10 oC), because
kinetic studies performed at 25 oC were compromised by the nature
of the compounds tested. This is
shown in Figure 1B, where the “overshoot” of long RT antagonist 27
happened before the t1
checkpoint of 20 min, which did not happen at 10 oC. A significant
correlation was also observed
between the antagonist affinities (Ki values) determined in
equilibrium displacement experiments
and their kinetic KD values derived from competition association
experiments (Figure 2B), despite
the differences in assay temperature (25 oC vs 10 oC).
Interestingly, the kinetic association rate
constants (kon) did not show any significant correlation with
affinity (Figure 2C), whilst the
dissociation rate constants (koff) had a fair correlation with
affinity (Figure 2D).
The representative long RT and short RT antagonists (27 and 5) were
selective for the hA3 receptor
when compared to other adenosine receptors (i.e. human adenosine A1
and A2A receptor, Table S1).
These two antagonists (27 and 5) with comparable association rate
constants but distinct
dissociation rate constants (or RTs) were further analyzed in a
[35S]GTPγS binding assay in which we
studied the (in)surmountable antagonism induced by the two
compounds (Figure 3).
Moreover, a kon-koff-KD “kinetic map” (Figure 4) was constructed
based on the compounds’ divergent
affinities (expressed as kinetic KD values) and kinetics
parameters, yielding a division of these
antagonists into three different sub-categories: antagonists that
show similar koff values (< 2-fold) but
due to differing kon values (> 28-fold) have different KD values
(~100-fold, Group A); antagonists that
display similar KD values (< 10-fold) despite showing divergent
koff and kon values (17-fold and 30-fold,
Group B); antagonists with similar kon values (< 5-fold), but
due to differing koff values (~290-fold)
have different KD values (> 110-fold, Group C). Additionally, we
applied molecular modeling to
compare the binding behavior in some molecular detail of several
antagonists with similar affinities
(2 vs 10; 31 vs 29 or 30) (Figure 5).
130
A B
C D
Figure 2: The correlations between the negative logarithm of the
human adenosine A3 receptor antagonists’ dissociation rates (pkoff)
and their kinetic rate index (KRI) (A), the human adenosine A3
receptor antagonists’ affinity (pKi) and their “kinetic KD” (pKD)
(B), association rate constants (logkon) (C), dissociation rate
constants (pkoff) (D). The central line corresponds to the linear
regression of the data, the dotted lines represent the 95%
confidence intervals for the regression. Data used in these plots
are detailed in Tables 1 - 3. Data are expressed as mean from at
least three independent experiments.
6 7 8 9 1 0
6
7
8
9
1
2
3
4
5
1
2
3
4
5
4 .5
5 .0
5 .5
6 .0
6 .5
7 .0
A. B.
C. D.
Figure 3: 2-Cl-IB-MECA-stimulated [35S] GTPγS binding to hA3R
stably expressed on CHO cell membranes (25 °C) in the absence or
presence of long-residence-time antagonist 27 (A and B, normalized
and combined, n ≥3), or short-residence-time antagonist 5 (C and D,
normalized and combined, n ≥3). Antagonist 27 (A), 5 (C) were
incubated for 60 min prior to the challenge of the hA3R agonist
2-Cl-IB-MECA, at a concentration ranging from 0.1 nM to 10 µM, for
another 30 min. Antagonist 27 (B), 5 (D) were coincubated with
2-Cl-IB-MECA, at the same concentration range, for 30 min. The
agonist curves were generated in the presence of increasing
concentrations of antagonists, namely 30-, 100-, 300-fold their
respective Ki values. Curves were fitted to a four parameter
logistic dose-response equation. Data is from at least three
independent experiments performed in duplicate, normalized
according to the maximal response (100%) produced by 2-Cl-IB-MECA
alone. The shift in agonist EC50 values was determined to perform
Schild analyses. Two-way ANOVA with Dunnett’s post-test was applied
for the comparison of Emax by agonist control, * p < 0.05, ** p
< 0.01, *** p < 0.001, **** p < 0.0001, **** p <
0.0001, ns for not significant.
Structure-Affinity Relationships (SAR) and Structure-Kinetics
relationships (SKR).
According to previous studies from our group,23, 24
methoxy-substitution at the C8 position (Table 1)
of the pyrido[2,1-f]purine-2,4-dione scaffold yielded selective
hA3R antagonists with good affinity
(3.2 nM for 1 as a reference compound). From our preliminary
studies, this methoxy-group appeared
important for slow dissociation (1 vs Compound S2 from Figure S1,
Table S2). Due to the nanomolar
affinity and close-to-unity KRI value of 1, it was treated as the
starting point of this SAR and SKR
study, having, on further analysis, an association rate constant of
(8.5 ± 1.2) x 105 M-1s-1 and a
-1 0 -9 -8 -7 -6 -5 -2 0
0
1 0 0
1 2 0
L o g M 2 -C l-IB -M E C A
[ 3
2 -C l-IB -M E C A
+ 3 0 K i c o m p o u n d 2 7
+ 1 0 0 K i c o m p o u n d 2 7
+ 3 0 0 K i c o m p o u n d 2 7
ns ns ns
0
1 0 0
1 2 0
L o g M 2 -C l-IB -M E C A
[ 3
2 -C l-IB -M E C A
+ 3 0 K i c o m p o u n d 2 7
*** ***
****
0
1 0 0
1 2 0
L o g M 2 -C l-IB -M E C A
[ 3
2 -C l-IB -M E C A
+ 3 0 K i c o m p o u n d 5
+ 1 0 0 K i c o m p o u n d 5
+ 3 0 0 K i c o m p o u n d 5
ns ns ns
0
1 0 0
1 2 0
L o g M 2 -C l-IB -M E C A
[ 3
2 -C l-IB -M E C A
+ 3 0 K i c o m p o u n d 5
+ 1 0 0 K i c o m p o u n d 5
+ 3 0 0 K i c o m p o u n d 5
ns ns ns
132
dissociation rate constant of (3.2 ± 0.02) x 10-4 s-1 (RT = 52
min). Next, we decided to investigate R1
substitutions (Table 2), beginning with antagonist 5 (R1 =
H).
The substitutions at R1 (Table 2).
Firstly, an increase in alkyl chain length was investigated,
indicating an elongated carbon chain had a
cumulative effect on KRI (5, 6, 7, 8, 10, 11), with the exception
of antagonist 9 (KRI values from 0.38
to 4.06). One could point to a possible correlation between
lipophilicities and dissociation rate
constants (and consequently RTs) to explain this trend (Figure
S2A). However, with all of the
antagonists kinetically characterized, no such correlation was
observed (Figure S2B). Therefore other
reasons should be taken into account as to why elongating the
carbon chain has such a profound
effect on the ligand’s dissociation rate. The role of membrane-drug
interactions in determining the
pharmacological profile is a possible reason, especially the role
long carbon tails have in such
interactions.28 Interesting to point out is the affinity of
antagonist 11 which, had a traditional lead
selection process taken place, would most likely have resulted in
the elimination of this compound
due the more favorable affinity and hydrophilic properties of its
shorter carbon chain counterparts
(8 or 9) (affinities: 6.8 nM vs 1.5 nM or 3.5 nM, KRI values: 4.06
vs 1.29 or 1.11). This would have
overlooked the efficacy this compound could offer due to its longer
residence time.
Secondly, the presence of a more rigid substitution of the R1 group
of saturated equivalents
(antagonists 1 and 8) led to antagonists 12 and 14, with similar
improvement in the affinity pairs (12
and 14: 5.9 nM and 1.4 nM; 1 and 8: 3.2 nM and 1.5 nM) and KRI
values (12 and 14: 0.72 and 1.23; 1
and 8: 0.99 and 1.29). Further rigidification with alkyne (13)
rather than alkene (12) maintained
affinities (4.3 nM vs 5.9 nM) and increased KRI values (1.20 vs
0.72). This alkyne could be the starting
point for a further study on “click-chemistry” for introducing
e.g., fluorescent tags.29-31
Thirdly, the introduction of a polar atom or group in antagonist 15
or 16, respectively, led to a
decrease in affinity compared to their non-polar counterpart 1 (23
nM or 81 nM vs 3.2 nM). The
133
changes in KRI values between antagonist 1 and its polar
counterparts 15 and 16 can be considered
minor (0.99 vs 0.70 and 1.04). Of note, by comparing affinities and
kinetic profiles of polar
antagonist 15 with its non-polar equivalent 1, we found the
polarity at the “lipophilic carbon chain”
resulted in slower association (kon of (4.3 ± 0.8) x 105 M-1s-1 vs
(8.5 ± 1.2) x 105 M-1s-1) but faster
dissociation (koff of (6.3 ± 0.7) x 10-4 s-1 vs (3.2 ± 0.02) x 10-4
s-1), with a concomitant decrease in
affinity (23 nM vs 3.2 nM).
Moreover, the bulkiness of the substituents was studied with
branched carbon side chains (17, 18,
19, 20 and 21) or aliphatic rings (2 and 22). As to the branched
carbon side chains, compound
affinities remained in the nanomolar range, while in terms of KRI
values, 2-carbon-linker branched
side chains (17 and 18) caused larger KRI values than those of
either their linear counterparts (8 and
9) or 3-carbon-linear branched side chains (19 and 20) (17: 1.64 vs
1.29 or 1.39; 18: 1.73 vs 1.11 or
0.95). Although the association rate constants of 17 and 18 were
similar to other antagonists in
Table 2, the dissociation rate constants suggest their branched
side chains have an extra “anchoring”
effect, compared with the linear counterparts. For example, the
koff of 18 with a 5-carbon branched
side chain was quite similar to 10 or 11 having a 6 or 7-carbon
linear side chain ((1.1 ± 0.4) x 10-4 s-1
vs (8.2 ± 1.3) x 10-5 s-1 or (6.2 ± 0.2) x 10-5 s-1). The presence
of a slightly less polar but larger silicon
atom (21) instead of carbon (18) made the KRI value decrease (1.36
vs 1.73), although the affinity
remained virtually the same (2.7 nM vs 3.5 nM).
Interestingly, another reported analogue (2)23 of compound 1, with
cyclopropylmethyl substitution
at the R1 group, led to unique kinetic parameters, i.e. a
combination of a fast association rate
constant ((2.8 ± 0.5) x 106 M-1s-1 vs (8.5 ± 1.2) x 105 M-1s-1) and
a slow dissociation rate constant ((6.0
± 1.7) x 10-5 s-1 vs (3.2 ± 0.02) x 10-4 s-1), although the
affinities of 2 and 1 were similar (1.0 ± 0.03 nM
vs 3.2 ± 0.1 nM, respectively). The RT of compound 2 was the
longest in Table 2 with 315 min. For
the antagonist with cyclobutylmethyl (22), affinity (2.7 nM vs 1.0
nM) and KRI value (1.48 vs 2.68)
were lower than for compound 2.
134
Although the dissociation rate constants of the antagonists in
Table 2 varied greatly depending on
the R1 substituent, the association rate constants were more
similar (within 5-fold). Association rate
constants are often reasoned to be caused by a diffusion limited
process whereby the collision rate
of ligand and receptor determines the rate of ligand-receptor
complex formation.32 When no
conformational changes are required for the receptor and ligand to
bind and when taking into
account the proportion of the receptor responsible for binding,
this sets the association rate
constant at observed limits of around 107 M-1s-1.33 As the
association rate constants for all R1
substituted compounds were slower than the diffusion limit by at
least 3.5 fold (2), we hypothesize
target engagement for R1 substituted antagonists is more hampered
than imposed by the diffusion
limit.
From Table 2 we learned that cyclopropylmethyl-substituted
antagonist 2 exhibited a kinetic profile
as a long RT compound whilst showing the affinity previously
reported.23 As a result, this compound
became the starting point for our exploration of the substitutions
(R2-group) on the aromatic ring.
Introduction of a non-polar alkyl substituent on antagonist 2’s
benzyl ring (24, 25, 26), resulted in a
decrease in KRI values (from 2.68 to 0.81), while slight variations
in affinity were observed. Then,
introduction of a polar methoxy substituent on antagonist 2’s
benzyl ring, led to mixed results with a
small decrease in RT at para-position, and a slight increase in RT
at meta-position in 28 (250 min vs
315 min) and 27 (376 min vs 315 min), respectively. In particular,
the long residence time for 27 in
combination with its sub-nanomolar affinity (0.38 nM), made this
compound stand out in the series.
Next, halogen substitutions on antagonist 2’s benzyl ring were
examined. Apparently, the position of
halogen substitution is important for affinity as para-substitution
in antagonist 30 and 32 yielded
similar affinity compared to 2 (1.2 nM vs 1.2 nM vs 1.0 nM). The
one compound with meta-
substitution, 29, showed a 5-fold decrease in affinity compared to
2 (4.9 nM vs 1.0 nM). Dichloro-
135
substituted compound 31 had the largest KRI value (3.12) among the
halogen-substituted
antagonists; the para-bromo substituted compound 32 was similar in
this respect to para-chloro
substituted 30 (1.19 vs 1.11). In a full competition association
experiment we determined the rate
constants for 31, and learned it had the longest RT of all
compounds kinetically characterized (391
min), concomitant with the slowest association rate constant of the
compounds kinetically
characterized ((1.0 ± 0.1) x 105 M-1s-1). Previous theoretical
studies have indicated the strength of
halogen bonding can be increased through the introduction of
electron withdrawing groups onto
halobenzenes.34 Such would be the case for 31 where the additional
chloro substituent forms a
stronger halogen bonding interaction with the R2 binding pocket.
Introducing a phenethyl (33) rather
than benzyl substituent (2) led to a decrease in affinity (7.7 nM
vs 1.0 nM), whilst the KRI value was
also strongly affected (1.09 vs 2.68). This observation parallels
our previous findings that the binding
pocket for the R2 substituent is of limited size.23
Functional Assay.
Following kinetic characterization, a long (27) and a short (5) RT
compound were chosen for
functional characterization in a [35S]GTPγS binding assay, also
because for these two compounds the
kon values were similar ((4.8 ± 0.2) x 105 M-1s-1 vs (5.3 ± 1.5) x
105 M-1s-1). This difference allowed a
possible link to be made between RTs and efficacies.
Pretreatment of hA3 receptor membranes with increasing
concentrations of the long RT antagonist
27, before stimulation by the A3 receptor agonist 2-Cl-IB-MECA,
induced insurmountable antagonism.
In other words, the 2-Cl-IB-MECA concentration-effect curves were
shifted to the right with a
concomitant decrease in the maximal response (Figure 3A).
Conversely, the short RT antagonist 5
displayed surmountable antagonism, shifting 2-Cl-IB-MECA’s curves
to the right without affecting its
maximum effect (Figure 3B).
136
Table 4. Functional activity of hA3 receptor antagonists from
[35S]GTPγS binding assays
Preincubation Coincubation
Mode of
antagonism Ligands
insurmountability
5
2.2 ± 1.4 6.8 ± 0.4 1.0 ± 0.3 7.2 ± 0.4 1.0 ± 0.2 Competitive
surmountability
b Obtained from Schild analyses.
c N.A.: not applicable.
In this experimental set-up, the Schild-slope of 5 generated from
Schild-plots was close to unity
(Table 4), and the compound’s pA2 value was comparable with its pKi
value (6.8 ± 0.4 vs 7.0 ± 0.02).
We also performed co-incubation experiments with these antagonists
in the presence of 2-Cl-IB-
MECA. In this experimental set-up, all antagonists produced a
rightward shift of the 2-Cl-IB-MECA
concentration-effect curves without a suppression of the maximal
response (Figure 3C and D).
Notably, the Schild-slopes of both long and short RT antagonists
(27 and 5) were close to unity (0.9 ±
0.2 for 27, 1.0 ± 0.2 for 5, Table 4). In addition, the pA2 value
of 5 was comparable with the result
from the pre-incubation condition (7.2 ± 0.4 vs 6.8 ± 0.4, Table
4), and the pA2 value of 27 was also in
agreement with its pKi value (8.9 ± 0.3 vs 9.4 ± 0.03).
137
Kinetic Map.
Using the association (kon) and dissociation (koff) rate constants
obtained from competition
association experiments (Tables 1-3), a kinetic map (Figure 4) was
constructed by plotting these
values on the y-axis and x-axis, respectively. The dashed diagonal
parallel lines represent the
kinetically derived KD values (KD = koff/kon). Out of this map
three subgroups emerged. Group A
represents compounds that exhibit similar koff values, but with
vastly different kon values. As a
consequence a diverse range of KD values was observed. Previous SKR
studies have primarily focused
on optimizing dissociation rates and RTs for predicting in-vivo
efficacy and creating a kinetically
favorable ligand. Yet recently, there has been greater
acknowledgment of the important role that
the association rate constants may play in determining the efficacy
of a drug as the result of
increased rebinding or increased drug-target selectivity.19 A
kinetic map would thus allow for the
selection of compounds with appropriate RTs whilst exploring the
role of association rate constants
in determining efficacy by choosing a rapidly or slowly associating
compound, i.e. 2 or 31 ((2.8 ± 0.5)
x 106 M-1s-1 vs (1.0 ± 0.1) x 105 M-1s-1). Group B displays ligands
that exhibit a narrow range of affinity
(KD: 0.1 nM - 1 nM), yet a wide range of koff values that result in
RTs ranging from 20 min to 391 min.
This information would have gone unnoticed in a traditional SAR
hit-to-lead approach and would
most likely have led to the selection of high affinity compounds
not in possession of a potentially
efficacy promoting long residence time. Thus, combining SAR with
SKR aspects in lead optimization
would allow the selection of not only potent but also long RT
compounds through the drug
development pipeline. Lastly group C represents compounds that
present similar kon values but due
to differing koff values show considerable differences in
affinities (KD). This illustrates the differences
that were observed in the binding kinetics of the R1 and R2
substituents, as group C mainly consists
of R1 substituents (non-cyclopropylmethyl substituents), whilst
group A mainly consists of R2
substituents (cyclopropylmethyl substituents). This difference also
suggests a different mode of
receptor-ligand interaction during the binding process of the two
ligand groups.
138
Figure 4: Kinetic map (y axis: kon in M-1·s-1, x axis: koff in s-1)
of all compounds that were kinetically characterized in this study.
kon and koff values were obtained through competition association
assays performed at the hA3R. The kinetically derived affinity (KD
= koff/kon) is represented through diagonal parallel lines. Group
A: compounds that show similar koff values but due to differing kon
values have different KD values. Group B: compounds that display
similar KD values despite showing divergent koff and kon values.
Group C: compounds with similar kon values, but due to differing
koff values have different KD values.
Altogether, the construction of a kinetic map allows for a more
detailed categorization of
compounds’ affinities as dictated by their kinetic rate constants.
In previous studies, such a
separation has explained the different therapeutic effects
molecules exhibit highlighting the benefits
of such an in-depth analysis.35, 36
Given the putative link between RT and clinical efficacy, it may be
postulated that the lack of hA3R
antagonists progressing from preclinical trials is due to
insufficient selection criteria employed in
these initial phases of hA3R drug screening. As previously
reported, hA3R antagonists are reasoned to
be beneficial in the treatment of Chronic Obstructive Pulmonary
Disease (COPD).37 For this
indication, a number of antagonists are available that act at the
muscarinic M3 receptor.38 For these
therapeutics their dosing regime and thus duration of action have
been linked to their RT. For
example, aclidinium which requires a twice daily dosing regimen,
exhibits a much shorter RT than
tiotropium that in turn requires only once daily dosing.16 This
extended duration of action that
1 0 - 4 .5 1 0 - 4 .0 1 0 - 3 .5 1 0 - 3 .0 1 0 - 2 .5 1 0 - 2
.0
1 0 5
1 0 6
1 0 7
)
A B
C
R 1 S u b s t i t u te d c o m p o u n d s
R 2 S u b s t i t u te d c o m p o u n d s
139
enables long-lasting efficacy and practical dosing regimens at the
muscarinic M3 receptor is thought
to be a beneficial feature in the treatment of chronic
illnesses.39, 40 As hA3R antagonists can be used
to treat chronic COPD but also a number of other chronic disorders,
we could imagine that
considering the ligand’s kinetic profile early in the drug
screening process, would reduce the
likelihood of failure due to insufficient efficacy in future
clinical trials. Perhaps when selecting hA3R
antagonists with a favorable long RT, i.e. the group A in the
kinetic map, will we see the therapeutic
potential of the hA3R fulfilled.
Computational studies.
Finally, we decided to further investigate the ligand-receptor
interactions using a homology model of
the adenosine A3 receptor, based on the crystal structure of the
adenosine A2A receptor (PDB:
4EIY)41. WaterMap calculations were applied to try and explain the
variance in kinetic profiles of
different ligands by unfavorable hydration42, 43.
Antagonist 2 (in black stick representation) was docked in the
homology model. As a first step it was
placed inside the transmembrane bundle, with the tricyclic ring
system surrounded by TM3, TM6
and EL2. Hydrogen bonding was constrained between the
amide-hydrogen (-NH2, δ+) from Asn2506.55
and the carbonyl-oxygen (-C=O, δ-) at the C4-position of the
pyrido[2,1-f]purine-2,4-dione scaffold
(Figure 5, left). In order to compare differences between the
ligands, an “apo” WaterMap of the hA3
receptor was generated. Hydration sites shown as red and orange
spheres represent positions
where “unstable” water molecules are found. Antagonist 10
(hexyl-substitution), with comparable
koff ((8.2 ± 1.3) x 10-5 s-1 vs (6.0 ± 1.7) x 10-5 s-1) to 2 but
10-fold slower kon ((2.3 ± 1.0) x 105 M-1s-1 vs
(2.8 ± 0.5) x 106 M-1s-1), was docked with two different binding
modes in the same binding site
(Figure 5, up-right, cyan and grey sticks). We found additional
unstable waters (8, 11, 22 in Figure 5,
up-right) surrounding the lipophilic substituents of the compounds,
which could be explained as
hindrance when the antagonist is associating with the binding
site.
140
Figure 5. Docking of antagonist 2 into the binding site of the
homology model of the adenosine A3 receptor based on the crystal
structure of the adenosine A2A receptor (PDB: 4EIY).41 Antagonist 2
is represented by black sticks, and residues within 5 Å of 2 are
visualized as orange sticks. The protein is represented by orange
ribbons. Ligand and residues atoms color code: red = oxygen, blue =
nitrogen, white = hydrogen. The overlay of consecutively numbered
hydration sites (colored spheres; for color code, see below) were
calculated by WaterMap (Left). Hydration sites shown as red and
orange spheres represent positions were “unstable” water molecules
can be found, which should be displaced by antagonist 2. White
spheres symbolize “stable” water molecules, which are in exchange
with the bulk solvent. Two different binding modes are represented
for antagonist 10 (cyan and grey sticks), which shows that the
flexible hexyl chain can displace different hydration sites (8 for
grey and 11 for cyan). For the key hydration sites (8, 11, 22, 32,
37) surrounding the lipophilic “tails”, calculated ΔG values (in
kcal/mol) with respect to bulk solvent are shown (Up-right).
Hydration sites 6, 39, 42 and 45 are proposed to be displaced by
the 3,4 di-chloro substituents of 31; calculated ΔG values (in
kcal/mol) with respect to bulk solvent are shown
(Down-right).
The same WaterMap was used to investigate the kinetic profile of
antagonist 31. Indeed, hydration
sites 6, 39, 42 and 45 are proposed to be displaced by the
3,4-dichloro substituent. Thus, both the
association and dissociation of 31 were slowed down by these
unstable waters. For the association
process, the lipophilic 3,4-dichloro moiety has difficulty in
approaching the occupied unstable
hydration sites ((1.0 ± 0.1) x 105 M-1s-1, slowest kon in the whole
series); the same lipophilic 3,4-
dichloro substituent seems to provide more stabilization to the
receptor-ligand complex, thus
hampering the dissociation process ((5.3 ± 1.5) x 10-5 s-1, slowest
koff in the whole series).
141
Interestingly, by removing a single chloro atom at either the 3- or
4- position on the benzyl-ring (30
or 29), association and dissociation rate constants became faster
by approximately 10-fold. Although
the differences in their kon and koff values were modest (2-3
fold), the unstable hydration sites may
prevent the 4-chloro-substituted antagonist 30 from reaching the
hydration sites 6, 39 and 42 that
interact with the 3-Cl substituent; consequently, both its
association and dissociation rate constants
were faster than of the 3-chloro-substituted counterpart 29 (kon:
(3.0 ± 0.3) x 106 M-1s-1 vs (8.2 ± 1.3)
x 105 M-1s-1; koff: (8.2 ± 0.2) x 10-4 s-1 vs (4.7 ± 0.7) x 10-4
s-1).
Conclusions
We have demonstrated that, next to affinity, additional knowledge
of target binding kinetics is useful
for selecting and developing new hA3R antagonists in the early
phase of drug discovery. By
introducing proper substituents at the N3 position or the N1 benzyl
ring of a series of
pyridopurinediones, divergences in kinetic profiles were observed,
while almost all compounds had
high and often similar affinity. Two representative ligands (5 and
27) were tested in [35S]GTPγS
binding assays, confirming the link between their RTs and their
(in)surmountable antagonism.
According to these findings, a kon-koff-KD kinetic map was
constructed and subsequently the
antagonists were divided into three sub-groups. Additionally, we
also performed a computational
modelling study that sheds light on the crucial interactions
(including with water molecules) for both
the association and dissociation kinetics of this family of
antagonists. It should be mentioned that
the kinetic parameters were derived at the hA3R, which may be
different in e.g., rodents used in
advanced animal models. Still, this study suggests that favorable
long RTs would be a proper
indicator in the development of hA3R antagonists for chronic
inflammatory conditions, e.g. COPD.
Experimental section
Chemistry. All solvents and reagents were purchased from commercial
sources and were of
analytical grade. Distilled water will be referred to as H2O. TLC
analysis was performed to monitor
142
the reactions, using Merck silica gel F254 plates. Grace Davison
Davisil silica column material (LC60A,
30–200 µm) was used to perform column chromatography. Microwave
reactions were performed in
an Emrys Optimizer (Biotage AB, formerly Personal Chemistry). 1H
and 13C NMR spectra were
recorded on a Bruker DMX-400 (400 MHz) spectrometer, using
tetramethylsilane as internal
standard. Chemical shifts are reported in δ (ppm) and the following
abbreviations are used: s,
singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet.
The analytical purity of the final
compounds is 95% or higher and was determined by high-performance
liquid chromatography
(HPLC) with a Phenomenex Gemini 3 µm C18 110A column (50 x 4.6 mm,
3 µm), measuring UV
absorbance at 254 nm. The sample preparation and HPLC method was as
follows: 0.3–0.6 mg of
compound was dissolved in 1 mL of a 1:1:1 mixture of
CH3CN/H2O/t-BuOH and eluted from the
column within 15 min at a flow rate of 1.3 mL/min. The elution
method was set up as follows: 1–4
min isocratic system of H2O/CH3CN/1% TFA in H2O, 80:10:10; from the
4th min a gradient was
applied from 80:10:10 to 0:90:10 within 9 min, followed by 1 min of
equilibration at 0:90:10 and 1
min at 80:10:10. Liquid chromatography–mass spectrometry (LC–MS)
analyses were performed
using a Thermo Finnigan Surveyor—LCQ Advantage Max LC–MS system and
a Gemini C18
Phenomenex column (50 x 4.6 mm, 3 µm). The elution method was set
up as follows: 1–4 min
isocratic system of H2O/CH3CN/1% TFA in H2O, 80:10:10; from the 4th
min a gradient was applied
from 80:10:10 to 0:90:10 within 9 min, followed by 1 min of
equilibration at 0:90:10 and 1 min at
80:10:10.
1-Benzyl-8-methoxy-1H,3H-pyrido[2,1-f]purine-2,4-dione (5.)24
6-Amino-1-benzyluracil (4) 25 (10.8 g,
49.7 mmol, 1.00 eq.) was suspended in CH3CN (370 mL).
N-bromosuccinimide (17.7 g, 99.4 mmol,
2.00 eq.) was added to the suspension and the mixture was heated at
80 C for 1 hour, after which
full conversion was shown by TLC (1:9 CH3OH/CH2Cl2 + 3%
triethylamine). Subsequently 4-
methoxypyridine (15.1 mL, 149.2 mL, 3.00 eq.) was added and the
mixture was heated at 80 C
during 10 hours. Full consumption of the bromo intermediate was
shown by TLC (1% CH3OH/CH2Cl2).
A precipitate was formed overnight at RT which was collected by
filtration and washed with diethyl
143
ether. This yielded the desired compound as a white solid (10.2 g,
31.6 mmol, 64%). 1H NMR
(400MHz, DMSO-d6) δ: 11.11 (s br, 1H), 8.72 (d, J = 7.2 Hz, 1H),
7.39-7.29 (m, 4H), 7.28-7.22 (m, 2H),
6.91 (dd, J = 7.2, 2.0 Hz, 1H), 5.19 (s, 2H), 3.89 (s, 3H) ppm. NMR
was according to literature data.24
General procedure for the preparation of N3-substitited
1-benzyl-8-methoxy-1H,3H-pyrido[2,1-
f]purine-2,4-diones (1, 2, 6-22).24 The compounds were synthesized
using the procedure described
by Priego et al.,24 but 5 eq. of the alkyl halide was used instead
of 1.5 eq. and the reaction mixture
was heated to 80 oC overnight in all cases. The pure compounds were
obtained by silica column
chromatography using a mixture of petroleum ether/ethyl acetate
(2:1) as eluent, if not otherwise
stated.
obtained by column chromatography using petroleum ether/ethyl
acetate (1:1) as eluent, yielding a
white solid 113 mg, 0.31 mmol, 60%. 1H NMR (400MHz, CDCl3) δ: 8.83
(d, J = 7.2 Hz, 1H), 7.54 (d, J =
7.2 Hz, 2H), 7.34-7.25 (m, 3H), 6.98 (d, J = 2.0 Hz, 1H), 6.74 (dd,
J = 7.6, 2.8 Hz, 1H), 5.37 (s, 2H), 4.02
(t, J = 7.6 Hz, 2H), 3.92 (s, 3H), 1.74 (sextet, J = 7.6 Hz, 2H),
0.99 (t, J = 7.6 Hz, 3H) ppm.24 MS [ESI+H]+:
calcd for C20H20N4O3, 364.15; found, 365.0.
1-Benzyl-3-(cyclopropylmethyl)-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione
(2).23 The pure
product was obtained by column chromatography using a mixture of 2%
CH3OH/CH2Cl2 as eluent,
yielding a white solid, 1.51 g, 3.98 mmol, 86%. 1H NMR (400MHz,
CDCl3) δ: 8.82 (d, J = 7.2 Hz, 1H),
7.54 (d, J = 7.2 Hz, 2H), 7.33-7.23 (m, 3H), 6.97 (d, J = 2.4 Hz,
1H), 6.73 (dd, J = 8.4, 2.4 Hz, 1H), 5.37
(s, 2H), 3.94 (d, J = 7.4 Hz, 2H), 3.92 (s, 3H), 1.35-1.25 (m, 1H),
0.47-0.44(m, 4H) ppm. MS [ESI+H]+:
calcd for C21H20N4O3, 376.15; found, 376.9.
1-Benzyl-8-methoxy-3-methylpyrido[2,1-f]purine-2,4(1H,3H)-dione
(6). Reaction was performed in a
sealed tube and 50 eq. of methyl iodide was used. The residue was
purified by silica column
chromatography eluting with a petroleum ether/ethyl acetate (1:1)
mixture, yielding a white solid,
144
25 mg, 0.074 mmol, 15%. 1H NMR (400MHz, CDCl3) δ: 8.83 (d, J = 7.6
Hz, 1H), 7.54 (d, J = 7.2 Hz, 2H),
7.33-7.24 (m, 3H), 6.99 (d, J = 2.4 Hz, 1H), 6.75 (dd, J = 7.6, 2.4
Hz, 1H), 5.37 (s, 2H), 3.93 (s, 3H), 3.45
(s, 3H) ppm. MS [ESI+H]+: calcd for C18H16N4O3, 336.12; found,
337.2.
1-Benzyl-3-ethyl-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione (7).
The pure product was obtained
by column chromatography using petroleum ether/ethyl acetate (1:1)
as eluent, yielding a white
solid, 58 mg, 0.17 mmol, 33%. 1H NMR (400MHz, CDCl3) δ: 8.83 (d, J
= 7.2 Hz, 1H), 7.54 (d, J = 7.2 Hz,
2H), 7.33-7.24 (m, 3H), 6.98 (d, J = 2.0 Hz, 1H), 6.74 (dd, J =
7.6, 2.8 Hz, 1H), 5.36 (s, 2H), 4.12 (q, J =
7.2 Hz, 2H), 3.92 (s, 3H), 1.28 (t, J = 7.2 Hz, 3H) ppm. MS
[ESI+H]+: calcd for C19H18N4O3, 350.14;
found, 351.0.
1-Benzyl-3-butyl-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione (8).
Purified by column
chromatography using petroleum ether/EtOAc (3:1), yielding a white
solid 10 mg, 0.026 mmol, 5%.
1H NMR (400MHz, CDCl3) δ: 8.83 (d, J = 7.6 Hz, 1H), 7.54 (d, J =
6.8 Hz, 2H), 7.24-7.33 (m, 3H), 6.98 (d,
J = 2.4 Hz, 1H), 6.74 (dd, J = 7.4, 2.6 Hz, 1H), 5.36 (s, 2H), 4.04
(t, J = 7.6 Hz, 2H), 3.93 (s, 3H), 1.70-
1.64 (m, 2H), 1.40 (sextet, J = 3.6 Hz, 2H), 0.95 (t, J = 7.2 Hz,
3H) ppm. MS [ESI+H]+: calcd for
C21H22N4O3, 378.17; found, 378.9.
1-Benzyl-8-methoxy-3-pentyl-1H,3H-pyrido[2,1-f]purine-2,4-dione
(9). White solid, 110 mg, 0.28
mmol, yield 56%. 1H NMR (400MHz, CDCl3) δ: 8.83 (d, J = 7.6 Hz,
1H), 7.56 (d, J = 7.2 Hz, 2H), 7.34-
7.25 (m, 3H), 6.97 (d, J = 2.0 Hz, 1H), 6.74 (dd, J = 7.6, 2.8 Hz,
1H), 5.37 (s, 2H), 4.05 (t, J = 7.6 Hz, 2H),
3.93 (s, 3H), 1.72-1.66 (m, 2H), 1.39-1.37 (m, 4H), 0.91 (t, J =
7.2 Hz, 3H) ppm. MS [ESI+H]+: calcd for
C22H24N4O3, 392.18; found, 393.1.
1-Benzyl-3-hexyl-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione
(10). White solid, 90 mg, 0.22
mmol, yield 44%. 1H NMR (400MHz, CDCl3) δ: 8.83 (d, J = 7.2 Hz,
1H), 7.55 (d, J = 6.8 Hz, 2H), 7.34-
7.25 (m, 3H), 6.98 (d, J = 2.0 Hz, 1H), 6.74 (dd, J = 7.6, 2.4 Hz,
1H), 5.37 (s, 2H), 4.05 (t, J = 7.6 Hz, 2H),
145
3.92 (s, 3H), 1.69 (pentet, J = 7.6 Hz, 2H), 1.40-1.26 (m, 6H),
0.89 (t, J = 7.2 Hz, 3H) ppm. MS [ESI+H]+:
calcd for C23H26N4O3, 406.20; found, 407.1.
1-Benzyl-3-heptyl-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione
(11). Column chromatography
using a mixture of petroleum ether/ethyl acetate (3:1) as eluent
yielded 131 mg of the pure product
as white solid, 0.31 mmol, 62%. 1H NMR (400MHz, CDCl3) 1H NMR
(400MHz, CDCl3) δ: 8.80 (d, J =
7.6 Hz, 1H), 7.54 (d, J = 6.8 Hz, 2H), 7.32-7.25 (m, 3H), 6.95 (d,
J = 2.4 Hz, 1H), 6.71 (dd, J = 7.2, 2.4 Hz,
1H), 5.35 (s, 2H), 4.03 (t, J = 7.6, 2H), 3.90 (s, 3H), 1.67
(pentet, J = 7.6 Hz, 2H), 1.38-1.26 (m, 8H),
0.87 (t, J = 7.2 Hz, 3H) ppm. MS [ESI+H]+: calcd for C24H48N4O3,
420.22; found, 421.2.
3-Allyl-1-benzyl-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione
(12). Purified by column
chromatography using petroleum ether/ethyl acetate (1:1) as eluent,
yielding a white solid, 73 mg,
0.20 mmol, 40%. 1H NMR (400MHz, CDCl3) δ: 8.82 (d, J = 7.6 Hz, 1H),
7.55 (d, J = 7.2 Hz, 2H), 7.34-
7.25 (m, 3H), 6.99 (d, J = 2.0 Hz, 1H), 6.75 (dd, J = 7.6, 2.4 Hz,
1H), 6.02-5.92 (m, 1H), 5.38 (s, 2H),
5.29 (dd, J = 17.2, 1.2 Hz, 1H), 5.20 (d, J = 10.0 Hz, 1H), 4.68
(d, J = 5.6 Hz, 2H), 3.93 (s, 3H) ppm. MS
[ESI+H]+: calcd for C20H18N4O3, 362.14; found, 363.0.
1-Benzyl-8-methoxy-3-(prop-2-yn-1-yl)pyrido[2,1-f]purine-2,4(1H,3H)-dione
(13). The pure product
was obtained by column chromatography using petroleum ether/ethyl
acetate (1:1) as eluent,
yielding a white solid, 60 mg, 0.17 mmol, 3%. 1H NMR (400MHz,
CDCl3) δ: 8.82 (d, J = 7.2 Hz, 1H),
7.57 (d, J = 6.8 Hz, 2H), 7.36-7.26 (m, 3H), 6.99 (d, J = 2.4 Hz,
1H), 6.76 (dd, J = 7.2, 2.4 Hz, 1H), 5.38
(s, 2H), 4.83 (d, J = 2.4 Hz, 2H), 3.94 (s, 3H), 2.19 (t, J = 2.4
Hz, 1H) ppm. MS [ESI+H]+: calcd for
C20H16N4O3, 360.12; found, 361.1.
1-Benzyl-3-(but-3-en-1-yl)-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione
(14). White solid, 18 mg,
0.05 mmol, yield 10%. 1H NMR (400MHz, CDCl3) δ: 8.84 (d, J = 7.6
Hz, 1H), 7.54 (d, J = 7.2 Hz, 2H),
7.35-7.28 (m, 3H), 7.00 (d, J = 2.4 Hz, 1H), 6.77 (dd, J = 7.6, 2.4
Hz, 1H), 5.91-5.84 (m, 1H), 5.39 (s,
146
2H), 5.08 (dd, J = 16.8, 1.2 Hz, 1H), 5.02 (d, J = 10.4, 1H), 4.15
(t, J = 7.2 Hz, 2H), 3.95 (s, 3H), 2.48 (q, J
= 7.2 Hz, 2H) ppm. MS [ESI+H]+: calcd for C21H20N4O3, 376.15;
found, 376.9.
1-Benzyl-8-methoxy-3-(2-methoxyethyl)-1H,3H-pyrido[2,1-f]purine-2,4-dione
(15). Purified by column
chromatography using a mixture of 2% CH3OH in CH2Cl2 yielded the
product as a white solid, 70 mg,
0.18 mmol, 37%. 1H NMR (400MHz, CDCl3) δ: 8.83 (d, J = 7.2 Hz, 1H),
7.54 (d, J = 6.8 Hz, 2H), 7.33-
7.24 (m, 3H), 6.98 (d, J = 2.4 Hz, 1H), 6.74 (dd, J = 7.6, 2.4 Hz,
1H), 5.36 (s, 2H), 4.30 (t, J = 6.0 Hz, 2H),
3.93 (s, 3H), 3.68 (t, J = 6.0 Hz, 2H), 3.36 (s, 3H) ppm. MS
[ESI+H]+: calcd for C20H20N4O3, 380.15;
found, 380.8.
column chromatography using a mixture of petroleum ether/EtOAc
(1:2), followed by
recrystallization from CH3OH/EtOAc. Yield: white solid, 106 mg,
0.28 mmol, 28%. 1H NMR (400MHz,
CDCl3) δ: 8.80 (d, J = 7.2 Hz, 1H), 7.53 (d, J = 7.6 Hz, 2H),
7.33-7.24 (m, 3H), 6.99 (d, J = 2.4 Hz, 1H),
6.76 (dd, J = 7.6, 2.4 Hz, 1H), 5.37 (s, 2H), 4.22 (t, J = 6.0 Hz,
2H), 3.93 (s, 3H), 3.53 (t, J = 5.6 Hz, 2H),
1.92 (pentet, J = 5.6 Hz, 2H) ppm. MS [ESI+H]+: calcd for
C20H20N4O3, 380.15; found, 380.9.
1-Benzyl-3-isobutyl-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione
(17). White solid, 0.08 mmol, 29
mg, yield 15%. 1H NMR (400MHz, CDCl3) δ: 8.86 (d, J = 7.2 Hz, 1H),
7.55 (d, J = 7.2 Hz, 2H), 7.33-7.28
(m, 3H), 7.00 (d, J = 2.0 Hz, 1H), 6.76 (dd, J = 7.6, 2.4 Hz, 1H),
5.39 (s, 2H), 3.95 (s, 3H), 3.92 (d, J = 7.6
Hz, 2H), 2.22 (nonet, J = 7.2 Hz, 1H), 0.97 (d, J = 6.8 Hz, 6H)
ppm. MS [ESI+H]+: calcd for C21H22N4O3,
378.17; found, 379.0.
1-Benzyl-8-methoxy-3-(3-neopentyl)-1H,3H-pyrido[2,1-f]purine-2,4-dione
(18). Purified by column
chromatography using a mixture of 1% CH3OH in CH2Cl2 as eluent,
yielding the product as a white
solid, 0.025 mmol, 20mg, 5%. 1H NMR (400MHz, CDCl3) δ: 8.84 (d, J =
7.2 Hz, 1H), 7.52 (d, J = 7.2 Hz,
2H), 7.32-7.25 (m, 3H), 6.98 (d, J = 2.4 Hz, 1H), 6.73 (dd, J =
7.2, 2.4 Hz, 1H), 5.37 (s, 2H), 3.99 (s, 2H),
3.92 (s, 3H), 0.98 (s, 9H) ppm. MS [ESI+H]+: calcd for C22H24N4O3,
392.18; found, 393.0.
147
1-Benzyl-3-isopentyl-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione
(19). Yield: white solid, 104 mg,
0.26 mmol, 53%. 1H NMR (400MHz, CDCl3) δ: 8.83 (d, J = 7.2 Hz, 1H),
7.53 (d, J = 7.2 Hz, 2H), 7.33-
7.24 (m, 3H), 6.98 (d, J = 2.4 Hz, 1H), 6.74 (dd, J = 7.6, 2.4 Hz,
1H), 5.36 (s, 2H), 4.08-4.04 (m, 2H),
3.93 (s, 3H), 1.69 (nonet, J = 6.8 Hz, 1H), 1.59-1.53 (m, 2H), 0.98
(d, J = 6.4 Hz, 6H) ppm. MS [ESI+H]+:
calcd for C22H24N4O3, 392.18; found, 393.1.
1-Benzyl-3-(3,3-dimethylbutyl)-8-methoxy-pyrido[2,1-f]purine-2,4(1H,3H)-dione
(20). Yield: white
solid, 81 mg, 0.20 mmol, 40%. 1NMR (400MHz, CDCl3) δ: 8.84 (d, J =
7.6 Hz, 1H), 7.55 (d, J = 7.2, 2H),
7.35-7.27 (m, 3H), 6.98 (d, J = 2.4 Hz, 1H), 6.74 (dd, J = 7.2, 2.4
Hz, 1H), 5.38 (s, 2H), 4.12-4.08 (m,
2H), 3.93 (s, 3H), 1.61-1.57 (m, 2H), 1.04 (s, 9H) ppm. MS
[ESI+H]+: calcd for C23H26N4O3, 406.20;
found, 407.1.
1-Benzyl-8-methoxy-3-((trimethylsilyl)methyl)pyrido[2,1-f]purine-2,4(1H,3H)-dione
(21). White solid,
137 mg, 0.34 mmol, yield 67%. 1H NMR (400MHz, CDCl3) δ: 8.84 (d, J
= 7.2 Hz, 1H), 7.50 (d, J = 6.8
Hz, 2H), 7.32-7.24 (m, 3H), 6.98 (d, J = 2.4 Hz, 1H), 6.73 (d, J =
7.2, 2.4 Hz, 1H), 5.39 (s, 2H), 3.93 (s,
3H), 3.64 (s, 2H), 0.08 (s, 9H) ppm. MS [ESI+H]+: calcd for
C21H24N4O3Si, 408.16; found, 409.2.
1-Benzyl-3-(cyclobutylmethyl)-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione
(22). White solid, 90
mg, 0.23 mmol, yield 46%. 1H NMR (400MHz, CDCl3) δ: 8.82 (d, J =
7.2 Hz, 1H), 7.53 (d, J = 7.2 Hz,
2H), 7.33-7.13 (m, 3H), 6.69 (d, J = 1.8 Hz, 1H), 6.73 (dd, J =
7.2, 2.4 Hz, 1H), 5.35 (s, 2H), 4.11 (d, J =
7.6 Hz, 2H), 3.92 (s, 3H), 2.83-2.72 (m, 1H), 2.05-1.95 (m, 2H),
1.90-1.79 (m, 4H) ppm. MS [ESI+H]+:
calcd for C22H22N4O3, 390.17; found, 391.2.
3-(Cyclopropylmethyl)-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione
(23).23 Prepared following a
slightly modified procedure described by Priego et al. In total 4
portions of 8 eq. of ammonium
formate (after 0, 2, 4 and 6 hours) and 3 portions of 0.15 eq. of
20% Pd(OH)2 (0, 4 and 6 hours) were
added, after which full conversion was reached after overnight
reflux visualized by TLC (3%
CH3OH/CH2Cl2). The reaction mixture was filtered over Celite and
the residue extracted 5 times with
148
hot DMF. The combined organic layer was concentrated in vacuo which
resulted in a quantitative
yield. 1H-NMR in accordance to data in literature.23
General procedure for the preparation of
N1-substitited-3-cyclopropylmethyl-8-methoxy-1H,3H-
pyrido[2,1-f]purine-2,4-diones (24-33).23 The compounds were
synthesized according to the
procedure described by Priego et al.2 Purification by silica column
chromatography using an eluent
mixture of petroleum ether/ethyl acetate (3:1) yielded the pure
final products.
3-(Cyclopropylmethyl)-8-methoxy-1-(3-methylbenzyl)pyrido[2,1-f]purine-2,4(1H,3H)-dione
(24).
White solid, 79 mg, 0.20 mmol, yield 57%. 1H NMR (400MHz, CDCl3) δ:
8.84 (d, J = 7.2 Hz, 1H), 7.36
(s, 2H), 7.22 (t, J = 7.6 Hz, 1H), 7.09 (d, J = 7.6 Hz, 1H), 6.99
(d, J = 2.4 Hz, 1H), 6.75 (dd, J = 7.2, 2.4 Hz,
1H), 5.36 (s, 2H), 3.97 (d, J = 7.2 Hz, 2H), 3.94 (s, 3H), 2.34 (s,
3H), 1.37-1.31 (m, 1H), 0.52-0.45 (m,
4H) ppm. MS [ESI+H]+: calcd for C22H22N4O3, 390.17; found,
391.0.
3-(Cyclopropylmethyl)-8-methoxy-1-(4-methylbenzyl)pyrido[2,1-f]purine-2,4(1H,3H)-dione
(25).23
3-(Cyclopropylmethyl)-1-(4-ethylbenzyl)-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione
(26). White
solid, 43 mg, 0.11 mmol, yield 21%. 1H NMR (400MHz, CDCl3) δ: 8.82
(d, J = 7.6 Hz, 1H), 7.48 (d, J =
8.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 6.98 (d, J = 2.0 Hz, 1H),
6.73 (dd, J = 7.2, 2.4 Hz, 1H), 5.34 (s, 2H),
3.95-3.93 (m, 5H), 2.60 (q, J = 7.6 Hz, 2H), 1.34-1.28 (m, 1H),
1.19 (t, J = 7.6 Hz, 3H), 0.50-0.42 (m, 4H)
ppm. MS [ESI+H]+: calcd for C23H24N4O3, 404.18; found, 405.2.
3-(Cyclopropylmethyl)
-8-methoxy-1-(3-methoxybenzyl)pyrido[2,1-f]purine-2,4(1H,3H)-dione
(27).
White solid, 20 mg, 0.049 mmol, yield 14%. 1H NMR (400MHz, CDCl3)
δ: 8.83 (d, J = 7.6 Hz, 1H), 7.23
(t, J = 8.0 Hz, 1H), 7.15-7.10 (m, 2H), 6.97 (d, J = 1.6 Hz, 1H),
6.80 (dd, J = 8.4, 2.4 Hz, 1H), 6.74 (dd, J =
7.6, 2.4 Hz, 1H), 5.35 (s, 2H), 3.96-3.92 (m, 5H), 3.77 (s, 3H),
1.35-1.28 (m, 1H), 0.50-0.45 (m, 4H)
ppm. MS [ESI+H]+: calcd for C22H22N4O4, 406.16; found, 406.9.
3-(Cyclopropylmethyl)-8-methoxy-1-(4-methoxybenzyl)pyrido[2,1-f]purine-2,4(1H,3H)-dione
(28).23
1-(3-Chlorobenzyl)-3-(cyclopropylmethyl)-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione
(29).23
1-(4-Chlorobenzyl)-3-(cyclopropylmethyl)-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione
(30).23
3-(Cyclopropylmethyl)-1-(3,4-dichlorobenzyl)-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione
(31).23
1-(4-Bromobenzyl)-3-(cyclopropylmethyl)-8-methoxypyrido[2,1-f]purine-2,4(1H,3H)-dione
(32). White
solid, 22 mg, 0.048 mmol, yield 14%. 1H NMR (400MHz, CDCl3) δ: 8.85
(d, J = 7.2 Hz, 1H), 7.46 (s, 4H),
6.99 (d, J = 1.6 Hz, 1H), 6.87 (dd, J = 7.2, 2.0 Hz, 1H), 5.33 (s,
2H), 3.99-3.85 (m, 5H), 1.34-1.24 (m,
1H), 0.50-0.44 (m, 4H) ppm. MS [ESI+H]+: calcd for C21H19N4O3,
454.06; found, 455.0.
3-(Cyclopropylmethyl)
-8-methoxy-1-phenylethylpyrido[2,1-f]purine-2,4(1H,3H)-dione (33).
White
solid, 112 mg, 0.29 mmol, yield 82%. 1H NMR (400MHz, CDCl3) δ: 8.81
(d, J = 7.2 Hz, 1H), 7.35-7.25
(m, 4H), 7.20 (t, J = 7.2 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.73
(dd, J = 7.2, 2.4 Hz, 1H), 4.40 (t, J = 8.0
Hz, 2H), 3.95-3.90 (m, 5H), 3.12 (t, J = 8.0 Hz, 2H), 1.32-1.23 (m,
1H), 0.49-0.40 (m, 4H) ppm. MS
[ESI+H]+: calcd for C22H22N4O3, 390.17; found, 391.0.
Biology. Chemicals and Reagents.
[3H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-
imidazo[2,1-i]-purin-5-one26 ([3H]34, specific activity 56
Ci·mmol-1) was a gift from Prof. C.E. Müller
(University of Bonn, Germany). Unlabeled 34 was purchased from
Tocris Ltd. (Abingdon, UK). 5’-N-
ethylcarboxamidoadenosine (NECA) was purchased from Sigma-Aldrich
(Steinheim, Germany).
Adenosine deaminase (ADA) was purchased from Boehringer Mannheim
(Mannheim, Germany).
Bicinchoninic acid (BCA) and BCA protein assay reagents were
purchased from Pierce Chemical
Company (Rockford, IL, USA). Chinese hamster ovary cells stably
expressing the human adenosine A3
receptor (CHOhA3) were a gift from Dr. K-N Klotz (University of
Würzburg, Germany). All other
chemicals were obtained from standard commercial sources and were
of analytical grade.
Cell Culture and Membrane Preparation. Chinese Hamster Ovary (CHO)
cells, stably expressing the
human adenosine A3 receptor (CHOhA3), were cultured and membranes
were prepared and stored
150
as previously described44. Protein determination was done through
use of the bicinchoninic acid
(BCA) method45.
Radioligand Displacement Assay. Membrane aliquots containing ~15 μg
of CHOhA3 protein were
incubated in a total volume of 100 μL assay buffer (50 mM Tris-HCl,
5 mM MgCl2, supplemented with
0.01% CHAPS and 1 mM EDTA, pH 7.4) at 25 °C for 120 min.
Displacement experiments were
performed using 6 concentrations of competing antagonist in the
presence of a final concentration
of ~10 nM [3H] 34. At this concentration, total radioligand binding
did not exceed 10% of that added
to prevent ligand depletion. Nonspecific binding (NSB) was
determined in the presence of 100 μM
NECA. Incubation was terminated by rapid filtration performed on
96-well GF/B filter plates (Perkin
Elmer, Groningen, the Netherlands), using a PerkinElmer
Filtermate-harvester (Perkin Elmer,
Groningen, the Netherlands). After drying the filter plate at 50 oC
for 30 min, the filter-bound
radioactivity was determined by scintillation spectrometry using
the 2450 MicroBeta2 Plate Counter
(Perkin Elmer, Boston, MA).
Radioligand association and dissociation assays. Association
experiments were performed by
incubating membrane aliquots containing ~15 μg of CHOhA3 membrane
in a total volume of 100 μL
of assay buffer at 10 or 25 °C with ~10 nM [3H] 34. The amount of
radioligand bound to the receptor
was measured at different time intervals during a total incubation
of 120 min. Dissociation
experiments were performed by preincubating membrane aliquots
containing ~15 μg of protein in a
total volume of 100 μL of assay buffer at 10 or 25 °C for 60 min.
After the preincubation, radioligand
dissociation was initiated by the addition of 5 μl 100 μM unlabeled
NECA. The amount of radioligand
still bound to the receptor was measured at various time intervals
for a total of 120 min to ensure
that full dissociation from hA3 receptor was reached. Incubations
were terminated and samples were
obtained as described under Radioligand Displacement Assay.
Radioligand Competition Association Assay. The binding kinetics of
unlabeled ligands were
quantified using the competition association assay based on the
theoretical framework by Motulsky
151
and Mahan.46 The competition association assay was initiated by
adding membrane aliquots (15
μg/well) at different time points for a total of 240 min to a total
volume of 100 μl of assay buffer at
10 °C or 25 °C with ~10 nM [3H] 34 in the absence or presence of a
single concentration of competing
hA3R antagonists (i.e. at their IC50 value). Incubations were
terminated and samples were obtained
as described under Radioligand Displacement Assay. The “dual-point”
competition association
assays were designed as described previously,27 where in this case
the two time points were selected
at 20 (t1) and 240 min (t2).
[35S] GTPγS Binding Assay. The assays were performed by incubating
15 µg of homogenized CHOhA3
membranes in a total volume of 80 µl assay buffer (50 mM Tris-HCl
buffer, 5 mM MgCl2, 1 mM EDTA,
0.05% BSA and 1 mM DTT, pH 7.4) supplemented with 1 µM GDP and 5 µg
saponin. The assays were
performed in a 96-well plate format, where DMSO stock solutions of
the compounds were added
using a HP D300 Digital Dispenser (Tecan, Männedorf, Switserland).
The final concentration of
organic solvent per assay point was ≤0.1%. In all cases, the basal
level of [35S] GTPγS binding was
measured in untreated membrane samples, whereas the maximal level
of [35S] GTPγS binding was
measured by treatment of the membranes with 10 μM 2-Cl-IBMECA. For
the insurmountability
experiments, membrane preparations were pre-incubated with or
without antagonists (30-, 100-,
300-fold Ki values) for 60 min at 25 oC, prior to the addition of
2-Cl-IBMECA (10 µM to 0.1 nM) and
20 µl [35S] GTPγS (final concentration ~0.3 nM), after which
incubation continued for another 30 min
at 25 oC. For the surmountability (control) experiments,
antagonists and 2-Cl-IBMECA were co-
incubated with [35S] GTPγS for 30 min at 25 oC. For all
experiments, incubations were terminated and
samples were obtained as described under Radioligand Displacement
Assay, by using GF/B filters
(Whatman International, Maidstone, UK).
Data Analysis. All experimental data were analyzed using the
nonlinear regression curve fitting
program GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego,
CA). From displacement assays,
IC50 values were obtained by non-linear regression analysis of the
displacement curves. The obtained
152
IC50 values were converted into Ki values using the Cheng-Prusoff
equation to determine the affinity
of the ligands.47 The observed association rates (kobs) derived
from both assays were obtained by
fitting association data using one phase exponential association.
The dissociation rates were
obtained by fitting dissociation data to a one phase exponential
decay model. The kobs values were
converted into association rate constants (kon) using the equation
kon = (kobs – koff)/[L], where [L] is
the amount of radioligand used for the association experiments. The
association and dissociation
rates were used to calculate the kinetic KD using the equation KD =
koff/kon. Association and
dissociation rate constants for unlabeled compounds were calculated
by fitting the data into the
competition association model using “kinetics of competitive
binding”:46
) )(
(
where k1 is the kon of the radioligand (M-1s-1), k2 is the koff of
the radioligand (s-1), L is the radioligand
concentration (nM), I is the concentration of the unlabeled
competitor (nM), X is the time (s) and Y is
the specific binding of the radioligand (DPM). The control curve
(without competitor) from
competition association assays generates the k1 value and the k2
value was obtained from
Radioligand association and dissociation assays. With that the k3,
k4 and Bmax can be calculated,
where k3 represents the kon (M-1s-1) of the unlabeled ligand, k4
stands for the koff (s-1)of the unlabeled
ligand and Bmax equals the total binding (DPM). All competition
association data were globally fitted.
The residence time (RT, in min) was calculated using the equation
RT = 1/(60*koff), as koff values are
expressed in s-1. [35S] GTPγS binding curves were analyzed by
nonlinear regression using “log
153
(agonist) vs response-variable slope” to obtain potency, inhibitory
potency or efficacy values of
agonists and inverse agonists/antagonists (EC50, IC50 or Emax,
respectively). In the (in)surmountability
assays, Gaddum/Schild EC50 shift equations were used to obtain
Schild-slopes and pA2 values;
statistical analysis of two-way ANOVA with Tukey’s post-test was
applied. All experimental values
obtained are means of at least three independent experiments
performed in duplicate, unless stated
otherwise. R2 and P values were calculated using the GraphPad Prism
linear regression analysis
function. LogP (log partition coefficient) values were calculated
using Chemdraw Professional 15.0
(Cambridge Soft, Perkin Elmer, Waltham Mass).
Computational studies. A ligand optimized homology model of the
hA3R was generated, following a
similar approach as has been used before48 and using the Maestro
software package (Schroedinger
Inc, New York). In short: first different homology models were
constructed based on the high
resolution crystal structure of the adenosine A2A receptor (PDB:
4EIY),41 and using a sequence
alignment from GPCRDB.49, 50 In the subsequent steps we iteratively
optimized the model using
Prime.51-53 During every step the best model was selected based on
enrichment (BEDROC-160.9 and
ROC). For this we used a set of 100 diverse antagonists from
ChEMBL54 obtained by “Cluster
Molecules”.55 We matched 50 decoys to every ligand ionization
state, using the DUD-e web service.56
The final model used here showed excellent enrichment
(BEDROC-160.9: 0.55 ROC: 0.80). We
introduced a long residence time ligand, 2, in the putative ligand
binding site using Induced fit
docking,57 with H-bond constraints on Asn2506.55. Based on this we
generated a WaterMap42, 43 of
the apo state of the receptor. Other ligands were docked using
core-constrained docking (using the
core of 2 as constraints). Figures were rendered using
PyMol.58
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Supporting information
The binding affinities of short RT antagonist (5) and long RT
antagonist (27) at human adenosine A1
and A2A receptors, the KRI values of pyrido[2,1-f]purine-2,4- dione
derivatives without methoxy-
substitution at C-8 position, the comparison with their
methoxy-substituted counterparts, and the
correlation between pkoff and Log P of the compounds.
1. Binding affinities of short RT antagonist (5) and long RT
antagonist (27) at human adenosine A1
and A2A receptors.
Binding affinities of antagonist 5 at hA1 and hA2A receptors have
been reported before,1 whilst
binding affinities of antagonist 27 at hA1 and hA2A receptors were
determined as described
previously.2, 3 Percentage displacement of antagonists (n=2,
average) or Ki ± SEM (nM, n=3) at human
adenosine A1 and A2A receptors are listed in Table S1.
Table S1. Binding affinities of short RT antagonist (5) and long RT
antagonist (27) at human
adenosine A1 and A2A receptors.
a Displacement of [3H]DPCPX from CHO cell membranes expressing the
human adenosine A1 receptor. Percentage displacement at 10 µM (n=2,
average) or Ki ± SEM (nM, n=3).
b Displacement of [3H]ZM241385 from HEK293 cell membranes
expressing the human adenosine A2A receptor. Percentage
displacement at 10 (5) or 1 µM (27, n=2, average),
c Published data.1
2. The KRI values of pyrido[2,1-f]purine-2,4-dione derivatives
without methoxy-substitution at C-8
position.
158
position were obtained as described under “the ‘dual-point’
competition association assays”.
Experiments were designed as described previously,4 where in this
case the two time points were
selected at 20 (t1) and 240 min (t2). The results are listed in
Table S2.
Table S2. Kinetic Parameters of Pyrido[2,1-f]purine-2,4-dione
Derivatives without Methoxy-
substitution at C-8 Position.
a KRI ± SEM (n = 3) or KRI (n = 2, individual estimates in
parentheses), obtained at 10 oC from dual point
competition association assays at 10 °C with [3H]34 on CHO cell
membranes stably expressing the hA3R.
Compound R1 KRIa
S1 CH2CH3 0.61
S6 CH2C3H5 1.01
(1.08,0.93)
159
3. The comparison between the KRI values of
pyrido[2,1-f]purine-2,4-dione derivatives without
methoxy-substitution at C-8 position and their methoxy-substituted
counterparts
The comparison is presented in Figure S1.
Figure S1: Comparison of KRI values of compounds with (red bars)
and without (blue bars) C-8 methoxy
substitution. KRI values obtained from dual point competition
association assay on CHO cells stably expressing
hA3R at 10 °C. Each data point is the average of two or three
independent experiments performed in duplicate.
4. The correlation between pkoff and LogP for antagonists.
LogP values were calculated using Chemdraw Professional 15.0
(Cambridge Soft, Perkin Elmer,
Waltham, MA, USA). R2 and P values were calculated using the
GraphPad Prism linear regression
analysis function. The correlations are presented in Figure
S2.
A. B.
Figure S2: Correlation between pkoff and LogP for antagonists with
an elongated carbon chain (1, 5, 9, 10 and
11) at the R1 position (A), or for all antagonists (B), obtained
from competition association assays.
S 1 7 S 2 1 S 3
1 7
S 4
1 2
S 5
1 3
1
2
3
4
5
1
2
3
4
5
160
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